Surfactant protein D for the treatment of disorders associated with lung injury

ABSTRACT

Surfactant protein D (SP-D) is a member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. Described herein are methods and compositions for the treatment of disorders associated with lung injury, including methods and compositions for the treatment of bronchopulmonary disorder (BPD).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/062,682 filed on Oct. 24, 2013, now U.S. Pat. No. 9,492,503 which is a continuation of U.S. application Ser. No. 13/366,144 filed on Feb. 3, 2012 which claims priority to U.S. Provisional Application Ser. No. 61/439,760, filed on Feb. 4, 2011, which are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under HL085610 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled AIRWY003C2SEQLIST, created Nov. 1, 2016, which is about 12 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

Surfactant protein D (SP-D) is a member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung.

SUMMARY OF THE INVENTION

Described herein are methods and compositions for the treatment of disorders associated with lung injury, including methods and compositions for the treatment of bronchopulmonary disorder (BPD).

In one embodiment, a method for reducing the risk of developing bronchopulmonary dysplasia (BPD) is provided, comprising administering a composition comprising recombinant human SP-D (rhSP-D) and a pulmonary surfactant to a subject in need thereof in an amount effective to reduce the risk of developing BPD. In some aspects, the BPD is associated with lung injury from mechanical ventilation. In some aspects, the composition further comprises at least one protein selected from the group consisting of surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), and fragments and mimics thereof. In some aspects, the pulmonary surfactant is a synthetic surfactant protein. In some aspects, the dose of the rhSP-D is about 0.1 mg to about 10 mg/kg body weight of the subject. In some aspects, the dose of the rhSP-D is about 2 mg/kg body weight. In some aspects, the total dose of the composition is about 100 mg/kg body weight of the subject. In some aspects, the composition is administered intratracheally. In some aspects, the method further comprises selecting a subject at risk for developing BPD prior to administering the composition to the subject. In some aspects, the subject is an infant.

In another embodiment, a method of reducing pulmonary injury associated with ventilation in a subject is provided, comprising administering recombinant human surfactant protein D (rhSP-D) and a pulmonary surfactant to the subject in an amount effective to reduce pulmonary injury associated with the ventilation. In some aspects, the ventilation is mechanical ventilation. In some aspects, the composition further comprises at least one protein selected from the group consisting of surfactant protein A (SP-A), surfactant protein B (SP-B), surfactant protein C (SP-C), and fragments and mimics thereof. In some aspects, the pulmonary surfactant is a synthetic surfactant protein. In some aspects, the dose of the rhSP-D is about 0.1 mg to about 10 mg/kg body weight of the subject. In some aspects, the dose of the rhSP-D is about 2 mg/kg body weight of the subject. In some aspects, the total dose of the composition is about 100 mg/kg body weight of the subject. In some aspects, the composition is introduced intratracheally. In some aspects, the method further comprises selecting a subject at risk for developing BPD prior to administering the composition to the subject. In some aspects, the subject is an infant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that treatment with recombinant human surfactant protein D (rhSP-D) does not alter lung physiology in premature lambs. Premature newborn lambs were resuscitated after birth by ventilation with a peak inspiratory pressure (PIP) of H₂O, resulting in a mean Pco₂ of 40 mm Hg (B) and a mean V_(T) of 11 ml/kg (C) for rhSP-D treated lambs and controls. Surfactant was given at 20 minutes of age and ventilation was changed to regulate V_(T) at 8 to 9 ml/kg (C), requiring a mean PIP of 27 cm H₂O (A) for rhSP-D treated lambs and controls.

FIG. 2 shows the effects of rhSP-D treatment on lung function. (A) The modified ventilation index (MVI) was calculated as peak inspiratory pressure×P_(CO2)×respiratory rate/1,000. Although not significant, MVI tends to be better (lower) for the group treated with rhSP-D (+rhSP-D) at later times. (B) Po₂/Fio₂ was higher in the +rhSP-D group compared with the control group (*P<0.01 by two-way repeated measures analysis of variance (ANOVA) (overall comparison of control versus+rhSP-D group)). Po₂/Fio₂ was significantly decreased after 210 minutes in the control group (P<0.05 vs. 18 min by one-way ANOVA).

FIG. 3 shows that treatment with rhSP-D does not alter pressure-volume curves or lung histology in premature lambs. (A) The deflation limbs of pressure-volume curves were not different between the rhSP-D treated lambs and controls. (B, C) Lung histology assessed after staining with hematoxylin and eosin was similar for both groups. Histology was typical of immature lung, including thickened alveolar septal walls and patchy atelectasis. More alveolar fluid was observed in control lambs than in lambs treated with rhSP-D (+rhSP-D). Scale bar: 100 μm.

FIG. 4 shows that treatment with rhSP-D decreases the number of inflammatory cells in BALF and decreases neutrophil elastase (NE) activity. (A) Increased total inflammatory cells and neutrophils in BALF induced by ventilation were suppressed by rhSP-D. (B) NE activity was assessed by a spectrophotometric assay using a chromogenic substrate specific for NE. Treatment with rhSP-D-containing SURVANTA® decreased NE activity (*P<0.05 versus the control group).

FIG. 5 shows that treatment with rhSP-D decreases the number of proinflammatory markers in lung homogenates. (A) Increased expression of interleukin-8 (IL-8) mRNA was significantly suppressed by rhSP-D treatment. Although not significant (due to the large variation in the control group), mean values of interleukin-6 (IL-6), interleukin-1β (IL-1β), keratinocyte-derived chemokine (KC), and monocyte chemotactic protein 1 (MCP1) were generally lower in the +rhSP-D group. (B) IL-8 protein in lung homogenates was significantly decreased by rhSP-D treatment (*P<0.05 versus the control group), while IL-1β was not influenced by rhSP-D treatment.

FIG. 6 shows that the addition of rhSP-D to surfactant increased resistance against surfactant inhibition. (A) Surface tension was measured by a captive bubble surfactometer. SURVANTA®+buffer had high surface activity, and minimum surface tension was low and was not influenced by addition of rhSP-D. Plasma protein inhibited the surface tension-lowering properties of SURVANTA®, and minimum surface tension was increased. The addition of rhSP-D rendered the SURVANTA® more resistant to plasma protein inhibition, with low minimum surface tension in the presence of plasma protein (n=3, *P<0.05 versus SURVANTA®+buffer with plasma). (B, C) Representative electron micrographs of SURVANTA® mixed with buffer or rhSP-D. Addition of rhSP-D changed the ultrastructure of SURVANTA® from simple lipid layers to the mixture of multiple lipid layers and lipid aggregates (n=3 per group). Scale bar: 500 nm.

DETAILED DESCRIPTION OF THE INVENTION

Premature newborns are routinely resuscitated by manual ventilation in the delivery room, followed by mechanical ventilation and surfactant treatment in the neonatal intensive care unit. The premature lung requires high inflating pressures and oxygen for adequate ventilation and oxygenation and is highly susceptible to injury because of its structural immaturity, surfactant deficiency, presence of fetal lung fluid, and immature immune system—factors that are likely to contribute to the development of the chronic lung disease bronchopulmonary dysplasia (BPD). Surfactant treatment is routinely given to very low birth weight (i.e., <1,500 g) preterm infants as early as possible after birth for the purpose of resuscitation in an effort to prevent and/or treat neonatal respiratory distress.

As described herein, rhSP-D can be added to resuscitation surfactant to improve surfactant distribution, minimize inhibition of surfactant function by leaked proteins, and prevent bronchopulmonary dysplasia (BPD)—a frequent consequence of the resuscitation process. Some embodiments relate to methods and compositions for the treatment of disorders associated with lung injury, including BPD. In one embodiment, recombinant human surfactant protein D (rhSP-D) is given in combination with a surfactant formulation to a mammal in need of treatment for a lung disorder. In some embodiments, the methods described herein reduce the risk of developing BPD. In some embodiments, the methods described herein reduce pulmonary injury associated with ventilation. In some embodiments, the methods described herein reduce pulmonary injury associated with the administration of oxygen, including high levels of oxygen. In some embodiments, the methods described herein reduce pulmonary injury associated with infection, such as intra-uterine infection.

In some embodiments, the mammal is a human. The human can be, e.g., an adult, a child, or an infant. In some embodiments, the infant is a newborn infant or a premature newborn infant. In some embodiments, the premature newborn infant is born at about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 weeks gestational age. In some embodiments, the newborn infant has a low birth weight. For example, in some embodiments, the birth weight of the newborn infant is less than about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 grams. In a preferred embodiment, the mammal is a premature newborn infant with respiratory disease (such as neonatal respiratory distress syndrome (nRDS)), or an infant at risk of developing or with symptoms of BPD.

In the embodiments described herein, a surfactant formulation can encompass one or more proteins, protein fragments, or mimics thereof. For example, in some embodiments, the surfactant formulation contains surfactant protein A (SP-A), and/or surfactant protein B (SP-B), and/or surfactant protein C (SP-C). In some embodiments, the surfactant formulation contains a combination of surfactant proteins, such as the combination of SP-B and SP-C. In some embodiments, the surfactant formulation contains a fragment of a surfactant protein. In some embodiments, the surfactant formulation contains lipids. For example, in some embodiments, the surfactant formulation contains dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the formulation contains DPPC and at least one of phosphatidylglycerol (PG) and phosphatidylinositol (PI). In a preferred embodiment, the surfactant formulation contains SP-B, SP-C, and DPPC.

In some embodiments, the surfactant formulation contains an animal derived surfactant. In some embodiments, the animal derived surfactant is a commercially available surfactant, such as ALVEOFACT®, CUROSURF®, INFASURF®, or SURVANTA®. In some embodiments, the animal derived surfactant is BLES®, SURFACEN®, or CLSE®. In some embodiments, the surfactant formulation contains a synthetic surfactant. In some embodiments, the synthetic surfactant is a commercially available synthetic surfactant, such as EXOSURF®, PUMACTANT®, SURFAXIN®, AEROSURF®, VENTICUTE®, or CHF 5633. In some embodiments, a combination treatment of rhSP-D and an animal surfactant is provided. In some of these embodiments, the animal surfactant contains at least one surfactant protein and at least one lipid. In some embodiments, a combination treatment of rhSP-D and a synthetic surfactant is provided. In some of these embodiments, the synthetic surfactant contains at least one recombinant protein, at least one surfactant protein fragment or mimic of a surfactant protein, and at least one lipid.

In some embodiments, the surfactant formulation contains a purified surfactant protein. In some embodiments, the surfactant formulation contains a recombinant protein. In some embodiments, the surfactant formulation contains a surfactant protein that is not SP-D. In some embodiments, the surfactant formulation does not contain a surfactant protein. In some embodiments, the surfactant formulation contains a lipoprotein complex. For example, in some embodiments, the surfactant formulation contains a phospholipoprotein complex.

The compositions described herein can be administered by any suitable route, including orally, intratracheally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. As used herein, the term “parenteral” includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion, or intraperitoneal administration. In a preferred embodiment, a composition described herein is administered intratracheally. In another preferred embodiment, a composition described herein is administered in an aerosolized form. For example, in one embodiment, rhSP-D and a surfactant formulation are administered as an aerosolized product that is administered by mask or continuous positive airway pressure (CPAP).

The compositions described herein can be administered as a single dose or in multiple doses. In some embodiments, the composition is administered once. In some embodiments, the composition is administered more than once. In a preferred embodiment, the composition is administered to a premature newborn infant in one or two doses. In some embodiments, rhSP-D and a surfactant formulation are each administered once per day. In some embodiments, rhSP-D and a surfactant formulation are administered together once per day. In some embodiments, rhSP-D and a surfactant formulation are administered together more than once per day.

In some embodiments, one or both of rhSP-D and a surfactant formulation is administered one, two, three, four, or more times per day. However, either or both can be administered less than once per day, e.g., about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

Suitable dose ranges vary, but in general, the rhSP-D can be administered in a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. In a preferred embodiment, the rhSP-D is provided in a dose of about 1 mg/kg to about 2 mg/kg body weight. In general, the surfactant formulation can be administered in a dose of about 25, 50, 75, 100, 125, 150, 175, 200, 300, 400, or 500 mg/kg body weight. In a preferred embodiment, the surfactant formulation is provided in a dose of about 100 mg/kg to about 200 mg/kg body weight. For example, in a preferred embodiment, the rhSP-D is provided in a dose of about 2 mg/kg body weight, and the surfactant formulation is provided in a dose of about 100 mg/kg body weight. In another preferred embodiment, the rhSP-D is provided in a dose of about 7 mg/kg body weight, and the surfactant formulation is provided in a dose of about 100 mg/kg body weight. In some embodiments, the dose of rhSP-D is about 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, or 5% of the total dose of a composition. For example, in a preferred embodiment, the dose of rhSP-D is 2 mg/kg body weight in a total dose of 100 mg/kg body weight (i.e., 2%) of rhSP-D and a surfactant formulation.

The selection of a particular dose may be based on the weight or identity of a mammal, the dose, and/or the dosing schedule of another co-administered compound. However, in some embodiments, it may be necessary to use doses outside the ranges described above. In some embodiments, the daily dosage of rhSP-D and a surfactant formulation are the same, and in some embodiments, the daily dosages vary. In some embodiments, the rhSP-D and surfactant formulation are administered together. In some embodiments, the rhSP-D and surfactant formulation are administered separately. In some embodiments, the daily dosage of a composition described herein is administered in a single dosage form. In some embodiments, the daily dosage of a composition described herein is administered in multiple dosage forms.

In some embodiments, at least one of rhSP-D and a surfactant formulation is administered in consistent daily dosages throughout the period of treatment. In some embodiments, at least one of rhSP-D and a surfactant formulation is administered in varying daily dosages during the period of treatment. In some of these embodiments, the daily dosages comprise increasing daily dosages over time. In some of these embodiments, the daily dosages comprise decreasing daily dosages over time.

In some embodiments, the dosage is adjusted so that the mammal maintains or exhibits reduced symptoms of a disorder. For example, in some embodiments, the dosage is adjusted so that a patient exhibits a reduction in symptoms of BPD. However, the dosage may also be adjusted by a treating physician based on a patient's particular needs. Further, the exact formulation, route of administration, and dosage can be chosen by a physician in view of the patient's condition.

In some embodiments, at least one of rhSP-D and a surfactant formulation is administered with varying frequency during treatment. In some of these embodiments, the varying frequency comprises a decreased frequency over time. For example, one or both of rhSP-D and the surfactant formulation can be initially administered more than once per day, followed by administration only once per day at a later point in treatment. In some embodiments, the daily dosage of at least one of rhSP-D and a surfactant formulation is consistent despite the varying frequency of administration.

In some embodiments, rhSP-D and a surfactant formulation are administered in a single pharmaceutical composition, such as a pharmaceutical composition comprising rhSP-D, a purified surfactant protein, a lipid, and a pharmaceutically acceptable carrier.

In some embodiments, administration is continued for a certain amount of time or until a particular outcome is achieved. For example, in some embodiments, administration of the compositions provided herein is continued for a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, 48, 72, 84, 96, 108, or 120 hours. In some embodiments, administration of rhSP-D and a surfactant formulation is continued until the reduction in symptoms of respiratory distress is stabilized for a period of at least about 12, 16, 20, 24, 36, 48, 72, 84, 96, 108, or 120 hours. In a preferred embodiment, symptoms of respiratory distress are stabilized for a period of about 72 hours to about 96 hours. In some embodiments, administration is continued for the duration of the life of a mammal. For example, in some embodiments, administration is continued daily, weekly, or monthly for the life of a human.

The compositions described herein may be accompanied by instructions for administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. Compositions formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Instructions and/or information may be present in a variety of forms, including printed information on a suitable medium or substrate (e.g., a piece or pieces of paper on which the information is printed), computer readable medium (e.g., diskette, CD, etc., on which the information has been recorded), or a website address that may be accessed via the internet. Printed information may, for example, be provided on a label associated with a drug product, on the container for a drug product, packaged with a drug product, or separately provided apart from a drug product, or provided in a manner in which a patient can independently obtain the information (e.g., a website). Printed information may also be provided to a medical caregiver involved in treatment of a patient.

The compositions described herein can be provided prior to, simultaneously with, or subsequent to ventilation and/or oxygen treatment. In some embodiments, the mammal receives ventilation and/or oxygen treatment for a period of time prior to receiving a composition. For example, in some embodiments, the mammal receives ventilation and/or oxygen treatment for about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 minutes prior to receiving a composition. In some embodiments, the mammal receives ventilation and/or oxygen treatment for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours prior to receiving a composition. In a preferred embodiment, a premature infant receives ventilation immediately following birth, and treatment with rhSP-D and a surfactant formulation within about 20 minutes of birth. For example, a premature infant can be intubated with an endotracheal tube and placed on a ventilator at birth, then receive rhSP-D and a surfactant formulation through the endotracheal tube about 20 minutes following birth. In some embodiments, the ventilation is manual ventilation. In some embodiments, the ventilation is mechanical ventilation. In some embodiments, the ventilation is both manual and mechanical. For example, in some embodiments, a premature infant is resuscitated by manual ventilation in the delivery room, followed by mechanical ventilation and treatment with rhSP-D and a surfactant formulation in the neonatal intensive care unit. In some embodiments, the mammal receives a composition about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 minutes prior to ventilation and/or oxygen treatment for a lung disorder. In some embodiments, the mammal receives a composition about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to treatment for a lung disorder. For example, in some embodiments, a patient with cystic fibrosis receives a composition within an hour of undergoing ventilation treatment. However, this timeframe can be adjusted by a treating physician based on a patient's particular needs.

In some embodiments, the mammal receives alternating treatment with a composition and ventilation and/or oxygen. For example, in some embodiments, the mammal receives ventilation, followed by a composition, followed by ventilation. In some embodiments, the mammal receives alternating and simultaneous treatment with a composition and ventilation and/or oxygen. For example, in some embodiments, the mammal receives a composition, followed by ventilation, followed by the composition and ventilation.

In some embodiments, the mammal is administered a composition described herein within a defined period of time following birth. In some embodiments, the mammal is administered a composition immediately following birth. In some embodiments, the mammal is administered a composition within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 minutes of birth.

The term “treatment” can include any intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with a disorder, as well as those in which the disorder is to be prevented. In some embodiments, the compositions described herein are useful to reduce the risk of developing BPD. In some embodiments, the compositions described herein are useful for reducing pulmonary inflammation associated with lung injury. In some embodiments, the compositions described herein are useful for reducing the symptoms of BPD resulting from lung injury.

The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues. The terms can apply to amino acid polymers in which one or more amino acid residue is an analog or mimic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be produced via several methods known in the art. For example, polypeptide products can be biochemically synthesized by employing standard solid phase techniques. Such methods include, but are not limited to, exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, and classical solution syntheses known to those of skill in the art. Polypeptides can also be generated using recombinant techniques known to those of skill in the art. For example, polypeptides can be synthesized by cloning a polynucleotide comprising the cDNA of a gene into an expression vector and culturing the cell harboring the vector to express the encoded polypeptide. In addition, polypeptides can be purified using methods known to those of skill in the art, including preparative high performance liquid chromatography. As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition. In some embodiments, a polypeptide is about 75%, 80%, 85%, 90%, 95%, or 99% pure. Polypeptides can also be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “protein,” “polypeptide,” and “peptide” include glycoproteins, as well as non-glycoproteins.

In some embodiments, the rhSP-D has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the rhSP-D has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the rhSP-D comprises the amino acid sequence of SEQ ID NO: 2 (NP_003010.4). In some embodiments, the rhSP-D comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 (precursor sequence of NP_003010.4). In some embodiments, the rhSP-D is encoded by the nucleic acid sequence of SEQ ID NO: 3 (NM_003019). In some embodiments, the rhSP-D is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some embodiments, the rhSP-D is expressed from a vector containing the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the rhSP-D is expressed from a vector containing a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. As will be appreciated by one of skill in the art, particular motifs and the degree of conservation between species provides guidance as to areas of rhSP-D that are important for structure and function (and are therefore not good candidates for varying from the sequences provided herein), and areas of variation that do not need to be conserved in the same manner.

The compositions described herein can include pharmaceutically acceptable carriers, such as adjuvants, excipients, and/or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the doses and concentrations employed. In some embodiments, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. For example, in some embodiments, compositions are pH adjusted with sodium bicarbonate. Examples of pharmaceutically acceptable carriers include, but are not limited to, adjuvants, lipids, preservatives, stabilizers, wetting agents, emulsifiers, and buffers. In some embodiments, the compositions contain a lipid. For example, in some embodiments, the compositions contain phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), sphingomyelin, tripalmitoylglycerol, palmitic acid, or mixtures thereof, In a preferred embodiment, compositions contain a rhSP-D, a surfactant protein, DPPC, and PI. Further, compositions can be prepared in solid form (including granules, powders or suppositories) or liquid form (e.g., solutions, suspensions, or emulsions). For example, in some embodiments, compositions are suspended in sodium chloride solution. In a preferred embodiment, the composition is in an aerosolized formulation.

EXAMPLE 1 rhSP-D Treatment in Premature Lambs

Premature lambs were delivered by cesarean section at 130 days' gestational age (GA) (full term is 150 days GA) and tracheostomized as previously described (Kramer B W, et al. Surfactant protein A recruits neutrophils into the lungs of ventilated pretemi lambs. Am J Respir Crit Care Med 2001; 163:158-165; Ikegami M, et al. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347). Premature newborn lambs were resuscitated with 100% O₂, a peak inspiratory pressure (PIP) of 40 cm H₂O, 4 cm H₂O positive end-expiratory pressure (PEEP), and a respiratory rate of 40/min using a pressure-limited ventilator (Sechrist Industries, Anaheim, Calif.). To avoid overstretch of the premature newborn infant lung during manual ventilation, the clinical resuscitation bag has a pressure relief valve set at 40 cm H₂O, and therefore PIP for resuscitation was limited to 40 cm H₂O.

Premature lambs at 130 days GA require surfactant treatment to survive. At 20 minutes of age, two groups of lambs were treated with SURVANTA® (Abbott Laboratories, Columbus, Ohio) mixed with rhSP-D (SEQ ID NO: 1) (+rhSP-D group) or buffer (control group) using two boluses for instillation (Ueda T, et al. Distribution of surfactant and ventilation in surfactant-treated preterm lambs. J Appl Physiol 1994; 76:45-55). rhSP-D was synthesized as previously described (Ikegami M, et al. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347; Ikegami M, et al. Surfactant protein-D and surfactant inhibit endotoxin induced pulmonary inflammation. Chest 2007; 132:1447-1454; Ikegami M, et al. Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes. J Appl Physiol 2009; 106:1545-1552). Seven milligrams of rhSP-D in 5 ml buffer (20 mM Tris, 200 mM NaCl, 1 mM ethylenediaminetetraacetic acid, pH 7.4) or 5 ml buffer alone were mixed with a clinical treatment dose of SURVANTA® (100 mg/4 ml/kg)—amounts that are similar to both SP-D and surfactant lipid pool sizes in the normal term newborn lung (Ikegami M, et al. Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes. J Appl Physiol 2009; 106:1545-1552; Ikegami M, Jobe A H. Surfactant metabolism. Semin Perinatol 1993; 17:233-240). After surfactant treatment, the PIP was decreased to regulate tidal volume (V_(T)) at 8 to 9 ml/kg (Bicore Monitoring Systems, Anaheim, Calif.), and F_(iO2) was adjusted to maintain a target P₀₂ of 100 to 150 mm Hg. Ventilatory rate, inspiratory time of 0.6 seconds, and PEEP were not changed.

A five hour study period was chosen to detect changes in proinflammatory cytokine mRNAs induced by initial ventilation (Naik A S, et al. Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung. Am J Respir Crit Care Med 2001; 164:494-498). After five hours, lambs were ventilated with F_(iO2)=1 for five minutes, then given 100 mg pentobarbital intravascularly, after which the endotracheal tube was clamped to permit oxygen absorption atelectasis (Ikegami M, et al. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347; Ikegami M, Jobe A. Postnatal lung inflammation increased by ventilation of preterm lambs exposed antenatally to E. coli endotoxin. Pediatr Res 2002; 52:356-362). After the thorax was opened, the deflation limb of pressure-volume curve was measured (Kramer B W, et al. Surfactant protein A recruits neutrophils into the lungs of ventilated preterm lambs. Am J Respir Crit Care Med 2001; 163:158-165; Ikegami M, et al. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347). Lung tissue of the right lower lobe was frozen in liquid nitrogen for RNA isolation and measurement of neutrophil elastase (NE) activity (Lowry O H, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275; Watterberg K L, et al. Secretory leukocyte protease inhibitor and lung inflammation in developing bronchopulmonary dysplasia. J Pediatr 1994; 125:264-269). NE is a potent serine proteinase, responsible for tissue destruction in the adult lung with emphysema (Shapiro S D, et al. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 2003; 163:2329-2335). In the preterm newborn lung, increased NE activity affects lung remodeling and increases alveolar epithelial apoptosis and the development of BPD (Watterberg K L, et al. Secretory leukocyte protease inhibitor and lung inflammation in developing bronchopulmonary dysplasia. J Pediatr 1994; 125:264-269; Yasumatsu R, et al. SERPINB1 upregulation is associated with in vivo complex formation with neutrophil elastase and cathepsin G in a baboon model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2006; 291:L619-L627). NE activity was assessed by a spectrophotometric assay using a chromogenic substrate specific for NE, N-methoxy-succinyl-Ala-Ala-Pro-Val pNA (Yasumatsu R, et al. SERPINB1 upregulation is associated with in vivo complex formation with neutrophil elastase and cathepsin G in a baboon model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2006; 291:L619-L627).

Sequences of primers for quantitative reverse transcriptase-polymerase chain reaction were: IL-8: 5′-TGGCCAGGATTCACGAGTTC (SEQ ID NO:5) and 5′-TCTGTGAGGTAGAAAGATGACTGAGATATT (SEQ ID NO:6); IL-6: 5′-GGAGGAAAAAGATGGATGCTTCCAA (SEQ ID NO:7) and 5′-CAGCAGTGGTTTTGATCAAGCAA (SEQ ID NO:8); IL-1β: 5′-GGCTCTCCACCTCCTCTCA (SEQ ID NO:9) and 5′-AGCTCATGCAGAACACCTT (SEQ ID NO:10); tumor necrosis factor (TNF)-α: 5′-GCCGGAATACCTGGACTATGC (SEQ ID NO:11) and 5′-CAGGGCGATGATCCCAAAGTAG (SEQ ID NO:12); keratinocyte-derived chemokine (KC): 5′-TGCCAGTGCCTGCAGAC (SEQ ID NO:13) and 5′-AGTGGCTATGACTTCGGTTTGG (SEQ ID NO:14); monocyte chemotactic protein (MCP1): 5′-CCCCGACTATCTGTTTCCACAAC (SEQ ID NO:15) and 5′-CCTGGAAGGGCTTCTGATCTG (SEQ ID NO:16); and ovine ribosomal protein L32 5′-GCAGAAGATTCAAGGGCCAGATC (SEQ ID NO:17) and 5′-GGTTTTCTTGTTGCTCCCGTAAC (SEQ ID NO:18).

Lung tissue of the right middle lobe was homogenized in 0.9% NaCl and supernatant after centrifugation at 1,000×g for 15 minutes and frozen for ELISA of proinflammatory cytokine proteins (Kramer B W, et al. Surfactant protein A recruits neutrophils into the lungs of ventilated preterm lambs. Am J Respir Crit Care Med 2001; 163:158-165; Ikegami M, et al. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347). BALF was recovered from the left lung (Ikegami M, et al. Whitsett J A. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347) for further analyses. Total proteins were analyzed (Lowry O H, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275) in the supernatant of BALF after 10 minutes of centrifugation at 284×g. The right upper lobe was inflation-fixed at 30 cm H₂O for morphology (Ikegami M, et al. Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes. J Appl Physiol 2009; 106:1545-1552). The amount of rhSP-D in aliquots of BALF was analyzed by ELISA (Ikegami M, et al. Whitsett J A. Intratracheal recombinant surfactant protein D prevents endotoxin shock in the newborn preterm lamb. Am J Respir Crit Care Med 2006; 173:1342-1347).

Results are given as means±SEM. Comparisons between +rhSP-D and control groups were made with two-tailed unpaired t tests. For multiple groups, one-way analysis of variance (ANOVA) followed by Bonferroni-Dunn test, or two-way repeated measures ANOVA were used. Significance was accepted at a P value<0.05.

EXAMPLE 2 Lung Function Following rhSP-D Treatment

Six control and six rhSP-D-treated lambs were studied. Sex (three male and three female per group), cord blood pH (7.36±0.04 [control], 7.36±0.05 [+SP-D]), body weight (3.0±0.3 [control], 3.0±0.1 kg [+SP-D]), and lung weight (116±15 [control], 115±5 g [+SP-D]) were similar between +rhSP-D and control groups. Blood pressure, heart rate, hematocrit, glucose, sodium, potassium, and calcium in the blood samples were recorded every 30 minutes and were normal throughout the study period (data not shown). Rectal temperature was maintained at the normal body temperature for sheep (38.5° C.) by means of heating pads, radiant heat, and plastic bodycovering wrap. Ventilation was regulated well for both groups. Lambs were resuscitated with PIP 40 cm H₂O for 20 minutes after birth (FIG. 1A), which resulted in mean Pco₂ of 40 mm Hg (FIG. 1B) and V_(T) of 11 ml/kg (FIG. 1C) for both groups. After surfactant treatment was given at 20 minutes of age, ventilation was changed to regulate V_(T) at 8 to 9 ml/kg (FIG. 1C) and required a mean PIP of 27 cm H₂O (FIG. 1A) for both groups. These results indicate that lung immaturity, as well as ventilatory stress used to support premature lambs, were comparable between the groups.

A modified ventilation index was calculated as PIP×Pco₂×respiratory rate/1,000 (Norden M A, et al. Predictors of survival for infants with congenital diaphragmatic hernia. J Pediatr Surg 1994; 29:1442-1446). Although it did not reach statistical significance, the mean modified ventilation index was better for the +rhSP-D group after 240 minutes (FIG. 2A). High Fi_(O2) (0.75-1.0) was used for both groups to maintain P₀₂ at the target. Premature lambs at this GA have patent ductus arteriosis, and P₀₂/F_(lo2) may not be directly associated with lung function. Nevertheless, P₀₂/F_(lo2) was higher in the +rhSP-D group than the control group (P<0.01 by two-way repeated measures ANOVA) (FIG. 2B). P02/Flo2 was significantly decreased after 210 minutes (P<0.05 by one-way ANOVA) in the control group.

The deflation limb of pressure-volume curves was not different between the groups (FIG. 3A). Likewise, lung morphology was similar for both groups, with typical findings consistent with immaturity, including thickened alveolar septal walls and patchy atelectasis. More fluid was noted in alveoli of the control lambs compared with the +rhSP-D lambs (FIGS. 3B and 3C).

EXAMPLE 3 Pulmonary Inflammation Following rhSP-D Treatment

Previous studies indicated a lack of inflammation as detected in BALF and lung tissue from 130-day GA lambs killed at delivery without ventilation (Naik A S, et al. Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preteim lamb lung. Am J Respir Crit Care Med 2001; 164:494-498; Ikegami M, Jobe A. Postnatal lung inflammation increased by ventilation of pre term lambs exposed antenatally to E. coli endotoxin. Pediatr Res 2002; 52:356-362).

Despite the use of a lung-protective ventilatory strategy, five hours of ventilation was associated with lung inflammation in the control lambs. In contrast, lung inflammation was decreased in all the lambs treated with rhSP-D. Inflammatory cells in the pellets collected by centrifugation were counted using trypan blue and differential cell counts were performed on the stained cytospin preparation (Kramer B W, et al. Surfactant protein A recruits neutrophils into the lungs of ventilated preterm lambs. Am J Respir Crit Care Med 2001; 163:158-165; Naik A S, et al. Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung. Am J Respir Crit Care Med 2001; 164:494-498). Total inflammatory cell numbers and neutrophils were significantly decreased by rhSP-D (FIG. 4A). Increased NE activity has been associated with the development of BPD (Watterberg K L, et al. Secretory leukocyte protease inhibitor and lung inflammation in developing bronchopulmonary dysplasia. J Pediatr 1994; 125:264-269; Yasumatsu R, et al. SERPINB1 upregulation is associated with in vivo complex formation with neutrophil elastase and cathepsin G in a baboon model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2006; 291:L619-L627). The addition of rhSP-D to SURVANTA® decreased NE activity in the lung (FIG. 4B). Expression of IL-8, IL-6, IL-1β, TNF-α, KC, and MCP1 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (FIG. 5A), and IL-8, IL-6, and IL-1β proteins in the supernatants of lung homogenates were measured by ELISA (FIG. 5B). Ovine ribosomal protein L32 was used as a reference RNA. Proinflammatory cytokine IL-8 (mRNA and protein), which plays a major role in neutrophil recruitment, was significantly decreased in the lung of rhSP-D-treated lambs. Although not statistically significant, mean IL-6 mRNA expression (P=0.06), and IL-6 protein (P=0.1) in the lung were lower in the +rhSP-D group. IL-1β protein and mRNA were not significantly influenced by rhSP-D treatment. Expression of TNF-α mRNA was similarly present at low levels in both groups (data not shown). KC, a functional homolog of IL-8, is critical for neutrophil recruitment and known to increase in ventilation-induced lung injury in adults (Belperio J A, et al. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 2002; 110:1703-1716). MCP1 possesses potent chemotactic activity for monocytes. Because of the large variation in lung inflammation in the control lambs, KC and MCP1 mRNA in the lung were not significantly different between the two groups, although mean levels were decreased by rhSP-D treatment.

The CD45 antibody recognizes the leukocyte common antigen and is present on cells of hematopoietic origin, except for erythroid cells and platelets. CD45-positive cells were isolated from BALF using magnetic cell separation (Miltenyi Biotech Inc., Auburn, Calif.) and CD14, CD11b, and CD44 were analyzed by flow cytometry (data not shown). CD14-positive cells were not detected in either group, suggesting that lung inflammation was not associated with infection. Both CD11b and CD44 influence vascular-to-tissue migration of neutrophils and monocytes to the sites of inflammation (Weirich E, et al. Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection. J Pediatr 1998; 132:445-451). Treatment with rhSP-D did not influence expression of CD11b or CD44, suggesting that suppression of neutrophil recruitment by rhSP-D in the lung was independent of changes in CD11 b and CD44.

After instillation of 7 mg of rhSP-D, 6.7±0.2 mg rhSP-D was recovered in BALF 4.7 hours after treatment. The slow clearance of exogenous rhSP-D from the lung is consistent with previous findings (Ikegami M, Jobe A H. Surfactant metabolism. Semin Perinatol 1993; 17:233-240), supporting the low rate of surfactant clearance in the preterm lung. Human SP-D was not detected in BALF from control lambs.

EXAMPLE 4 Increased Resistance Against Surfactant Inhibition Following rhSP-D Treatment

Surface tension was measured by captive bubble surfactometer (Schoel M, et al. The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations. Biochim Biophys Acta 1994; 1200:281-290) on 3 μL of samples containing 15 μg/μL SURVANTA® and 2% rhSP-D or buffer in the presence or absence of surfactant inhibitor (21 μg/μL plasma protein) (Ikegami M, et al. Characteristics of surfactant from SP-A deficient mice. Am J Physiol Lung Cell Mol Physiol 1998; 275:L247-L25). This amount of plasma protein relative to SURVANTA® was 30% lower than the concentration that is known to inhibit the activity of SURVANTA® in the ventilated premature newborn lamb lung in vivo (Wada K, et al. Tidal volume effects on surfactant treatment responses with the initiation of ventilation in preterm lambs. J Appl Physiol 1997; 83:1054-1061). The influence of rhSP-D on the ultrastructure of SURVANTA® was studied as previously described (Schmiedl A, et al. Influence of plasma and inflammatory proteins on the ultrastructure of exogenous surfactant. J Electron Microsc (Tokyo) 2004; 53:407-416).

Minimum surface tension of SURVANTA® with (+rhSP-D) or without rhSP-D (+buffer) in the presence or absence of a surfactant inhibitor (plasma protein) was measured with a captive bubble surfactometer (FIG. 6A). The minimum surface tension was low with or without rhSP-D—consistent with the high surface activity of SURVANTA® and similarity of lung function and pressure-volume curves seen in both +rhSP-D-treated and control lambs. Immediately after mixing with plasma, surfactant mixtures were applied to the bubble. Plasma proteins inhibited the surface tension-lowering properties of SURVANTA®, with the minimum surface tension being increased to greater than 15 mN/m. The addition of 2% rhSP-D to SURVANTA® rendered the SURVANTA® more resistant to plasma protein inhibition, the minimum surface tension remaining low in the presence of plasma proteins. Because SP-D influences surfactant ultrastructure in the alveolus by causing lysis of surfactant lipid layers (Ikegami M, et al. Surfactant protein-D regulates the postnatal maturation of pulmonary surfactant lipid pool sizes. J Appl Physiol 2009; 106:1545-1552), ultrastructure of the surfactant mixtures used for treatment was assessed. The simple lipid layers formed by SURVANTA® (FIG. 6B) were changed by the addition of rhSP-D, causing the formation of lipid aggregates and multilayers (FIG. 6C). These changes in the ultrastructure of SURVANTA® caused by rhSP-D may be related to its resistance to inhibition of surface activity by plasma protein. Although proteins in BALF in both the control group (60±7 mg/kg) and +rhSP-D group (57±11 mg/kg) were threefold higher than that in non-ventilated premature lambs seen in previous studies (Naik A S, et al. Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung. Am J Respir Crit Care Med 2001; 164:494-498; Ikegami M, Jobe A. Postnatal lung inflammation increased by ventilation of pretemi lambs exposed antenatally to E. coli endotoxin. Pediatr Res 2002; 52:356-362), they were not high enough to inhibit the function of the large amount of SURVANTA® given to the lambs. Inhibition of surfactant function by plasma protein occurs when alveolar proteins are increased above 200 mg/kg (Wada K, et al. Tidal volume effects on surfactant treatment responses with the initiation of ventilation in preterm lambs. J Appl Physiol 1997; 83:1054-1061).

EXAMPLE 5 rhSP-D and Animal Surfactant Treatment in a Premature Infant

rhSP-D and natural animal surfactants, including SURVANTA®, INFASURF®, and CUROSURF®, are administered to premature infants. The combinations of rhSP-D and each of the natural animal surfactants are tested clinically against the natural surfactants alone (i.e., a test for superior effects). The premature infants are assessed clinically for the prevention and/or treatment of neonatal respiratory distress syndrome (RDS), and for the prevention of bronchopulmonary dysplasia (as these conditions are defined by one of skill in the art at the time of the study). Study endpoints include the percentage of infants recovering from RDS and the incidence of BPD in the study population.

EXAMPLE 6 rhSP-D and Synthetic Surfactant Treatment in a Premature Infant

rhSP-D and a synthetic surfactant, with or without any component of surfactant protein, are administered to premature infants. The combination of rhSP-D and the synthetic surfactant is tested clinically against the synthetic surfactant alone (i.e., a test for superiority), and/or a natural surfactant alone (i.e., a test for superiority), and/or the combination of rhSP-D and a natural surfactant (i.e., a test for equivalence or superiority). The premature infants are assessed clinically for the prevention and/or treatment of neonatal respiratory distress syndrome (RDS), and for the prevention of bronchopulmonary dysplasia (as these conditions are defined by one of skill in the art at the time of the study). Study endpoints include the percentage of infants recovering from RDS and the incidence of BPD in the study population. 

What is claimed is:
 1. A method of treating a subject having a pulmonary disorder selected from the group consisting of a pulmonary injury resulting from mechanical ventilation, neonatal respiratory distress syndrome, a pulmonary injury resulting from administration of oxygen, and a bronchopulmonary dysplasia, comprising administering an effective amount of a surfactant in combination with an effective amount of surfactant protein D (SP-D) to the subject, wherein the effective amount of the surfactant is less than the effective amount of the surfactant in the absence of the SP-D, and is sufficient to treat the pulmonary disorder.
 2. The method of claim 1, wherein the pulmonary disorder is a pulmonary injury resulting from administration of oxygen.
 3. The method of claim 1, wherein the pulmonary disorder is a pulmonary injury resulting from mechanical ventilation.
 4. The method of claim 1, wherein the amount of the SP-D) is sufficient to reduce the activity of an endogenous surfactant inhibitor in a lung of the subject.
 5. The method of claim 4, wherein the endogenous surfactant inhibitor comprises a plasma protein.
 6. The method of claim 1, wherein the amount of the SP-D is sufficient to reduce the activity of an endogenous surfactant inhibitor on the surface tension lowering properties of the surfactant in a lung of the subject.
 7. The method of claim 1, wherein the amount of the SP-D is sufficient to reduce the surface tension lowering properties of the surfactant in a lung of the subject by about 4-fold compared to the surface tension lowering properties of the surfactant in the presence of an endogenous surfactant inhibitor in a lung of the subject and in the absence of the SP-D.
 8. The method of claim 1, wherein the SP-D is recombinant human SP-D or a functional fragment thereof.
 9. The method of claim 1, wherein the surfactant and SP-D) are administered in a single pharmaceutical composition.
 10. The method of claim 9, wherein the pharmaceutical composition comprises about 2% SP-D.
 11. The method of claim 9, wherein the pharmaceutical composition comprises a phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol.
 12. The method of claim 9, wherein the surfactant and SP-D are administered in an aerosolized form.
 13. The method of claim 9, wherein the surfactant and SP-D are administered in a dry powder form.
 14. The method of claim 1, wherein the dose of the SP-D is about 0.1 mg/kg to about 10 mg/kg body weight of the subject.
 15. The method of claim 1, wherein the dose of the SP-D is about 2 mg/kg body weight of the subject.
 16. The method of claim 1, wherein the surfactant is an animal product.
 17. The method of claim 1, wherein the surfactant is bovine surfactant.
 18. The method of claim 1, wherein the surfactant is synthetic surfactant.
 19. The method of claim 1, wherein the subject is a neonate.
 20. The method of claim 1, wherein the subject is a preterm neonate. 